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. 2023 Feb 24;24(5):4511.
doi: 10.3390/ijms24054511.

General Trends of the Camelidae Antibody VHHs Domain Dynamics

Akhila Melarkode Vattekatte  1   2 , Julien Diharce  1 , Joseph Rebehmed  3 , Frédéric Cadet  2   4 , Fabrice Gardebien  2 , Catherine Etchebest  1 , Alexandre G de Brevern  1   2
Affiliations

General Trends of the Camelidae Antibody VHHs Domain Dynamics

Akhila Melarkode Vattekatte et al. Int J Mol Sci. .

Abstract

Conformational flexibility plays an essential role in antibodies' functional and structural stability. They facilitate and determine the strength of antigen-antibody interactions. Camelidae express an interesting subtype of single-chain antibody, named Heavy Chain only Antibody. They have only one N-terminal Variable domain (VHH) per chain, composed of Frameworks (FRs) and Complementarity Determining regions (CDRs) like their VH and VL counterparts in IgG. Even when expressed independently, VHH domains display excellent solubility and (thermo)stability, which helps them to retain their impressive interaction capabilities. Sequence and structural features of VHH domains contributing to these abilities have already been studied compared to classical antibodies. To have the broadest view and understand the changes in dynamics of these macromolecules, large-scale molecular dynamics simulations for a large number of non-redundant VHH structures have been performed for the first time. This analysis reveals the most prevalent movements in these domains. It reveals the four main classes of VHHs dynamics. Diverse local changes were observed in CDRs with various intensities. Similarly, different types of constraints were observed in CDRs, while FRs close to CDRs were sometimes primarily impacted. This study sheds light on the changes in flexibility in different regions of VHH that may impact their in silico design.

Keywords: Protein Blocks; antibody; disorder; flexibility; mobility; molecular dynamics simulation; nanobody; single-chain antibody; structural alphabet; sybody.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Sequence and structure characteristics in VHH dataset. Conservation of (A) amino acid residues, (B) secondary structures and (C) Protein Blocks. The four Framework regions are delineated in each figure.
Figure 2
Figure 2
Flexibility metrics at each residue position. (A) Representation of normalised Cα B-factors; the x-axis is the residue position in the MSA and the y-axis the normalised Cα B-factor values. (B) Representation of Cα RMSF values. (C) Representation of Neq values. The three CDR regions are highlighted using three red-coloured regions at the bottom of the plots. The average values of each metric are shown in dotted red lines.
Figure 3
Figure 3
Tendencies of flexibility metrics at each residue position. Mean and standard deviation of (A) normalised Cα B-factors, (B) Cα RMSF and (C) Neq. Occurrence is shown as a histogram. CDR positions are shown as ref lines.
Figure 4
Figure 4
Representation of mean values of flexibility metrics onto a 3D structure of a VHH. (A) coloured FRs and CDRs, (B) mean normalised Cα B-factor values, (C) mean Cα RMSF values and (D) mean Neq values of all VHHs.
Figure 5
Figure 5
Hierarchical clustering of VHH trajectories using RMSF. The different RMSF clusters are demarcated using coloured boxes, and their cluster number is marked accordingly.
Figure 6
Figure 6
Representation of mean values of flexibility metrics onto a 3D structure of a VHH from each cluster. (A–D) RMSF cluster1, (E–H) RMSF cluster 2, (I–L) RMSF cluster 3, (M–P) and RMSF cluster 4, with (A,E,I,M) are coloured coded FRs and CDRs, (B,F,J,N) mean normalised Cα B-factors, (C,G,K,O) mean Cα RMSF values, and (D,H,L,P) mean Neq values.
Figure 7
Figure 7
Local backbone diversity at the light of PBs. (A) PB map of all VHH trajectories aligned according to MSA, (B) Neq values (red line—all VHH trajectories, sky blue—VHH trajectories belonging to RMSF cluster 1, green—VHH trajectories belonging to RMSF cluster 2, purple—VHH trajectories belonging to RMSF cluster 3 and pink—VHH trajectories belonging to RMSF cluster 4), PBs map of VHH trajectories belonging to (C) from RMSF cluster 1, (D) from RMSF cluster 2, (E) from RMSF cluster 3 and (F) from RMSF cluster 4. The x-axis represents the residue positions, and the y-axis represents the types of PBs or the Neq.
Figure 8
Figure 8
PBs’ differences in terms between each RMSF cluster. (A) ΔPB (with RMSF cluster 1 in sky blue, with RMSF cluster 2 in green, with RMSF cluster 3 in purple and with RMSF cluster 4 in pink); CDRs are shown with red line. 3D visualisation on structures of ΔPB values for (B) RMSF cluster 1, (C) RMSF cluster 2, (D) RMSF cluster 3, and (E) RMSF cluster 4.
Figure 9
Figure 9
Mean of Distance between CDRs termini in each VHH trajectory. (A) CDR1, (B) CDR2 and (C) CDR3 termini.
Figure 10
Figure 10
Representation of backbone conformations from trajectories clustered using average ΔPB. (A,C,E,G) are PB maps of the four ΔPB clusters, (B,D,F,H) are the visualisation of the structures.
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